Vpr and vpx are two viral accessory genes that are expressed in primate lentiviruses, which are required for viral replication and disease progression in vivo. Vpr has two well-known activities in vitro: arrest of cell cycle in G2 phase and enhancement of viral replication in monocyte-derived macrophages. Sharing ~25% homology with Vpr, Vpx only has the viral enhancement activity. Although both Vpr and Vpx enhance HIV-1 replication, different mechanisms are involved. While Vpx counteracts the antiretroviral activity of SAMHD1, the mechanism of Vpr enhancement of viral replication is still unknown. Recently, we reported a potent HIV-1 restriction in the human CD4+ T cell line CEM.NKR (NKR), which was naturally isolated from the human T lymphoblastoid cell line CEM. Although NKR cells express both CD4 and CXCR4, HIV-1 replication is severely restricted from the 2nd round of replication. From the original NKR cells, we isolated three types of clones that show different levels of HIV-1 resistance: non-permissive (NP), semi-permissive (SP), and permissive (P). We then compared wild-type (WT) and Vpr-deficient ( Vpr) HIV-1 replication in these cells. In non-permissive cells, both WT and Vpr viruses were unable to replicate. Notably, a treatment with arsenic trioxide (As2O3) increased the WT virus replication by almost 1000-fold, but did not promote the Vpr virus replication. Similarly, although the WT virus could replicate in the semi-permissive and permissive cells, the Vpr virus replication was completely inhibited in the semi-permissive cells and significantly delayed in the permissive cells. These results suggest that Vpr is absolutely required for HIV-1 replication in the non-permissive and semi-permissive cells. Thus, we have identified Vpr-specific HIV-1 non-permissive human CD4+ T cell line, which represents an important progress in the Vpr field. Our objective is to decipher the mechanism of how Vpr enhances HIV-1 replication in NKR cells. Our hypothesis is that NKR cells may express a Vpr-sensitive restriction factor to block viral replication, or lack a Vpr-like positive factor to support viral replication. Our rationale is that these NKR cells provide a relevant model system for studying Vpr function, which will be useful for further characterization of Vpr activity in vivo. We propose three specific aims: 1) Delineate how Vpr enhances HIV-1 replication in NKR cells; 2) Identify the host factor in NKR cells that is responsible for Vpr- dependent HIV-1 replication; 3) Elucidate the relevance of HIV-1 restriction in NKR cells for HIV-1 biology. We will define the enigmatic role of Vpr in viral life cycle. The discovered new mechanism will be likely translated into innovative tools for antiretroviral therapy.